Drug-resistant Mycobacterium tuberculosis (Mtb) is an increasing threat to global health. A number of innovative genetic studies have identified genes and pathways that are essential to Mtb survival; however, these discoveries have produced few new validated drug targets and target-inhibitors. Similarly, recent studies have identified hundreds of novel hits active against Mtb using whole-cell screening; yet, very few promising drug-candidates have resulted from this work. The problem has been to find good matches between viable drug targets and compounds with known whole-cell activity. It is becoming increasingly apparent that not all essential metabolic processes represent good drug targets. However, years of drug development efforts have established cell wall biosynthesis, protein synthesis, and DNA supercoiling as critical cellular processes that do contain excellent targets for antibacterials. In fact, some inhibitors of these pathways are in clinical use as anti-B drugs. Inhibition of respiration comprises a fourth druggable pathway in Mtb, as demonstrated by the new respiration inhibitor bedaquiline. We propose to discover and develop inhibitors that target these druggable processes. We have already developed a screen that broadly detects cell wall biosynthesis inhibitors, and proved its effectiveness for specifically identifying new classes of compounds with that inhibit the cell wall. Our screening/discovery approach can also be adapted to identify inhibitors that are specific to other druggable cellular processes. Here, we propose to fully characterize the remaining hits from our cell wall inhibitor screen, expand our screening approach to uncover new inhibitors and novel targets in the druggable processes of protein synthesis, DNA supercoiling and respiration, validate each target, and develop selected hits into optimized drug leads. For the R21 phase we will: 1) Identify and validate the targets of promising new hit compounds already identified in our cell wall biosynthesis inhibitor screen. 2) Discover new hit chemical classes that inhibit the highly vulnerable processes of protein synthesis, DNA supercoiling and respiration in Mtb, using our novel whole cell promoter-reporter screening approach. The cellular targets of each promising hit will then be determined using whole-genome sequencing of resistant mutants. For the R33 phase we will select at least 10 of the most active hits across a range of targets and: 3) Validate the putative target of each hit wit biochemical and genetic studies. Then, further investigate the mode of action of each hit by studying the metabolic consequences within Mtb upon treatment with each hit, using a cutting edge metabolomic assay. 5) Finally, we will perform hit-to-lead optimization of at least six compounds.